CN104307208B - A kind of hematoblastic method of enriching and purifying - Google Patents
A kind of hematoblastic method of enriching and purifying Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/19—Platelets; Megacaryocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/64—Animal cells
Abstract
The invention discloses a kind of hematoblastic method of enriching and purifying, add anticoagulant in whole blood, 1000rpm is centrifuged 10 minutes, drawing red blood cell layer above section, add normal saline in residue red blood cell layer, 1000rpm is centrifuged 10 minutes, taking red blood cell layer above section, both are mixed to get enriched substance C；Enriched substance C adds erythrocyte cracked liquid, it is centrifuged 20 minutes with 1500rpm after standing half an hour after mixing, collect supernatant A and middle white batt layer A, supernatant A is centrifuged 10 minutes with 1500rpm, collects white layer B bottom it, mixes with middle white batt layer A, add normal saline, 1500rpm is centrifuged 10 minutes, collects middle white layer C, and gleanings is the platelet of enrichment method.The present invention adds blood plasma and broken erythrocytic purification process on the basis of centrifugal enrichment, can remove erythrocyte completely, is effectively improved platelet rich purity, reduces leukocyte and red blood cell contamination.
Description
Technical field:
The invention belongs to field of biomedicine technology, relate to one and be applicable to include that people is dynamic with experiment
Thing is in the hematoblastic centrifugal preparation method of interior different genera high-purity.
Background technology:
Platelet (platelet) is the akaryote in mammalian, less than erythrocyte and
Leukocyte, in addition to can providing cohesion, moreover it is possible to by intracellular α stopping blooding when
The degranulation of grain discharges a large amount of cell growth factor, including platelet-derived growth factor
(PDGF), transforming growth factor-β (TGF-β), IGF-1 (IGF-1),
Fibroblast growth factor (FGF), VEGF (VEGF) etc., can
The division growth of induction osteocyte and collage synthesis, promote reparation and the reconstruction (Marx of osseous tissue
RE,Implant Dent,2001,10(4):225-228).Wherein, PDGF can promote that skeletonization is thin
The chemotactic of born of the same parents, propagation, increase the synthesis capability of collagen protein；Before TGF-β has stimulation skeletonization
Somatic cell and osteoblastic chemotactic and propagation, suppression osteoclast formation and the work of bone resorption
With；IGF-1 can increase osteoblast vigor, promote that cartilage generates with bone matrix；VEGF can
Promote that new vessels is formed, the beneficially supply of focal zone nutrient substance, moreover it is possible to directly act on
Osteoblast and chondrocyte, strengthen its migrate, increment and cell viability (Bouletreau PJ,
et al.,Plast Reconstr Surg,2002,110(1):139-148；Maeda S,et al.,
EMOB J,2004,23(3):552-563；Trippel SB,Clin Orthop Relat Res.1998.
(355Suppl):301-313；Ferrara N,et al.,Nat Med.2003.9(6):669-676).
Platelet can be extracted from patient self whole blood, plays therapeutical effect, have after enriched process
Abundance, draws materials conveniently, separates the features such as simple, be widely used in orthopaedics, the department of stomatology,
The fields such as sports medical science.Existing platelet craft preparation method is platelet rich plasma (PRP)
Method, obtains PRP for experimentation and clinical treatment.
PRP is that whole blood passes through the platelet concentrate being centrifugally separating to obtain, and its preparation principle is profit
Different by various composition sedimentation velocity in blood, by centrifugal, blood is layered.Whole blood is through low speed
Centrifugal can be divided into three layers, upper strata is platelet poor plasma layer (PPP), middle for rich platelet
Plasma layer (PRP), lower floor are red blood cell layer, take intermediate layer can get high enrichment containing blood
Platelet-poor plasma.It is generally believed that PC is 1～3 × 10 in human whole blood5In/ml, PRP
PC should reach more than 4 times of Whole blood platelet concentration, the somatomedin contained is dense
Degree also should more than 3 times (Marx et al., Oral Surg Oral Med Oral Pathol Oral
Radiol Endod,1998,85(6):638；Landesberg et al.,J Oral Maxillofac Surg,
2000,58(3):297；Dugrillon et al.,Int J Oral Maxillofac Surg,2002,
31(6):615.).Existing PRP preparation method once centrifuging and secondary centrifuging method.Public
The centrifuging recognized is Anitua method, is that 10～20ml whole bloods are added 5ml containing anticoagulant
Centrifuge tube in, be centrifuged 6 minutes with 160g, upper strata is PPP, and lower floor is erythrocyte, two
A thinnest pale yellow chromatograph seen from layer intersection, i.e. PRP layer；Discard upper strata (PPP) 1ml,
Sucked away upper plasma, to lower floor's (red blood cell layer) 1～2mm, obtains PRP (Anitua, Int
J Oral Maxillofac Implants,1999,14(4):529-535.).Once it is centrifuged with Anitua
Method can obtain about 1.2mlPRP from 5ml whole blood every time.The secondary centrifuging method generally acknowledged has
Landesberg method, Petrungaro method and Aghaloo method, the difference of three be centrifugal force and
The difference of centrifugation time.As Landesberg method 200g for the first time is centrifuged 10 minutes, suction pipe
Draw whole supernatant (PPP+PRP) to 3mm under interface, move to another centrifuge tube,
After balance, second time 200g is centrifuged 10 minutes, draws about 3/4 supernatant (PPP) and discards,
Residue shakes up and is PRP；Petrungaro method for the first time 1500g is centrifuged 6 minutes, for the second time
1000g is centrifuged 6 minutes；Aghaloo method 215g for the first time is centrifuged 10 minutes, 863g for the second time
Centrifugal 10 minutes (Landesberg et al., J Oral Maxillofac Surg, 2000,
58(3):297-300；Petrungaro,Compend Contin Educ Dent,2001,22(9):
729-732；Aghaloo et al.,J Oral Maxillofac Surg,2002,60(10):
1176-1181.).The platelet count of above-mentioned three kinds of secondary centrifuging methods compares with recovery rate of blood platelet
Understanding, the platelet count that Aghaloo method obtains is most, the response rate the highest (Zhang Changqing and Yuan Ting,
Shanghai science tech publishing house, 2011, p32-33.).
Although the PRP that concentration is up to standard can be prepared at present, but its technology not yet full maturity.Nothing
Discuss a centrifuging or secondary centrifuging method all exists shortcomings, greatly limit it in experiment
Room and clinical application: 1. lack quality control system and the unified standard generally acknowledged, it is impossible to ensure
Prepare repeatability and the biological stability of PRP every time；2. platelet and somatomedin pick-up rate
Relatively low with purity, still there are a large amount of leukocyte and red blood cell contamination, there is potential risk；③
Cannot avoid untoward reaction that plasma residence causes (Brittinghan and Chaplin, JAMA,
1957,165(7):819-825.).Therefore, existing PRP preparation method still haves much room for improvement.
Bad kidney-Yang etc. (CN 102367435B, Chinese Patent Application No. 201110344464.0,
Denomination of invention: the preparation of people's platelet rich plasma and in human mesenchymal stem cell separation and Culture
Application) disclose the preparation method of a kind of people's platelet rich plasma, products therefrom fills for the human world
The separation of matter stem cell, cultivation have comparatively ideal effect.The method is collected with secondary centrifuging method
In whole blood platelet and carry out≤0 DEG C and be not less than normal temperature condition multigelation process, go
Except precipitation obtains platelet rich plasma.Multigelation can make plaque rupture, discharges a large amount of cell
Somatomedin.Therefore the main component of this invention acquisition is actual is the platelet rich in somatomedin
Lysate, finally removes settling step and has removed all cells residual, i.e. this product does not exist blood
Platelet.Zheng Qiujian etc. (CN 103505910A, Chinese Patent Application No. 201310480780.X,
Denomination of invention: a kind of method that a centrifuging prepares platelet rich plasma) disclose a kind of one
Secondary centrifuging prepares the method for platelet rich plasma, is similar to previously mentioned Anitua method.
(CN 103505911A, Chinese Patent Application No. 201310480851.6, the invention such as woods flood
Title: a kind of method preparing platelet rich plasma with manual secondary centrifuging method) and Lin Zhuoheng
Deng (CN 102755770A, Chinese Patent Application No. 201210267055.X, denomination of invention:
The extracting method of platelet rich plasma and the platelet rich plasma of extraction) individually disclose two kind two
Secondary centrifuging prepares the method for platelet rich plasma, and difference is centrifugal rotational speed and time, similar
In methods such as previously mentioned Landesberg.
Summary of the invention:
The present invention provides a kind of people and the manual preparation method of animal blood platelet enriching and purifying, especially
It is on the basis of centrifugal enrichment, add blood plasma and broken erythrocytic purification process.The method
In operation, it is not necessary to deliberately avoid extracting erythrocyte, but can remove erythrocyte completely,
There is higher operability with repeatable than existing manual platelet rich plasma preparation method
Property.Platelet rich purity can be effectively improved by the application of the present invention, reduce leukocyte and red
Cell contamination, avoid the potential adverse effect of blood plasma, it is intended to solve existing manual method and prepare PRP
Concentration or purity is the highest, leukocyte and red blood cell contamination, plasma residence and quality unstable etc.
Defect problem.
The technical solution used in the present invention is:
A kind of hematoblastic method of enriching and purifying, said method comprising the steps of:
(1) whole blood adds anticoagulant, be centrifuged 10 minutes with 1000rpm, after centrifugal end,
Whole blood is divided into three layers: upper strata be Platelet poor layer, middle level be rich platelet layer (tunica albuginea layer),
Lower floor is red blood cell layer, draws red blood cell layer above section, obtains enriched substance A；Residue
Red blood cell layer adds normal saline, then is centrifuged 10 minutes with 1000rpm, take red carefully
Born of the same parents' layer above section, obtains enriched substance B, and enriched substance B mixes with enriched substance A,
To enriched substance C；
(2) in enriched substance C, add erythrocyte cracked liquid, stand after mixing after half an hour with
1500rpm is centrifuged 20 minutes, collects supernatant A and middle white batt layer A respectively,
Supernatant A is centrifuged 10 minutes with 1500rpm, collects white layer B bottom it, with in
Between white flock layer A mixing, add normal saline, be centrifuged 10 points with 1500rpm
Clock, abandons supernatant B, collects middle white layer C, avoids the red residual in bottom during collection,
Gleanings is the platelet of enrichment method.
In described step (1), described anticoagulant is preferably sodium citrate, heparin or hirudin,
More preferably sodium citrate.
Described anticoagulant can add in whole blood in the way of the vacuum test tube containing anticoagulant, this
It is to well known to a person skilled in the art blood sampling anticoagulant methods.
Described anticoagulant is generally 1:9 with the volume ratio of whole blood, and the volume of described anticoagulant is
Refer to the volume of anticoagulant solution.
Described sodium citrate addition in whole blood is according to the body of liquor sodii citratis Yu whole blood
Long-pending ratio determines for 1:9, and the mass concentration of described liquor sodii citratis is 3.2%～3.8% (phase
When in 0.109mol/L～0.129mol/L).This ratio is (blue with containing sodium citrate vacuum test tube
Lid) consistent, therefore the present invention is easy to clinical blood specimen collection.
The concentration of described heparin is generally 15IU/mL.
The concentration of described hirudin is generally 0.2mg/mL.
In described step (1), whole blood is carried out two times centrifugal, it is therefore an objective to discard in whole blood
Major part erythrocyte, and fully collect platelet component.For separation platelet as much as possible
And erythrocyte, the present invention through early stage exploration discovery, with 1000rpm be centrifuged 10 minutes be
Good condition.
In described step (1), residue red blood cell layer adds normal saline, normal saline master
Playing the scattered effect of dilution, the addition of normal saline is generally the 0.2～1 of volume of whole blood
Times.
In described step (2), described erythrocyte cracked liquid is that aqueous ammonium chloride solution, lymph are thin
Born of the same parents separate liquid or other commercializations or the erythrocyte cracked liquid prepared voluntarily.
In described step (2), described aqueous ammonium chloride solution is 1:2 with the volume ratio of enriched substance C,
The mass concentration of described aqueous ammonium chloride solution is 1.0%.
Described lymphocyte separation medium can buy acquisition, according to the operation instruction of commodity on market
Carry out operating.Lymphocyte separation medium used in the embodiment of the present invention is purchased from Shanghai Suo Lai
Precious bio tech ltd, lymphocyte separation medium is 1:1 with the volume ratio of enriched substance C.
In described step (2), the purpose adding erythrocyte cracked liquid is red in order to remove residue
Cell, and assemble platelet by centrifugal；Purpose supernatant A being centrifuged again is the most
Collection platelet；The last purpose that mixes with normal saline and be centrifuged be in order to remove blood plasma and
Bib remains, and concentrates and purifies platelet further.The present invention through early stage exploration discovery,
Being centrifuged with 1500rpm is above-mentioned optimum condition.
In described step (2), collect white layer B bottom it, mix with middle white layer A,
Adding normal saline, normal saline primarily serves the scattered effect of dilution, adding of normal saline
Enter amount and be generally 0.2～1 times of volume of whole blood.
Further, the method for the invention preferably sequentially includes the following steps:
(1) adding anticoagulant in whole blood, anticoagulant is 1:9 with the volume ratio of whole blood, with
1000rpm is centrifuged 10 minutes, after centrifugal end, draws red blood cell layer above section, obtains
Enriched substance A；Residue red blood cell layer adds normal saline, then is centrifuged 10 points with 1000rpm
Clock, takes red blood cell layer above section, obtains enriched substance B, and enriched substance B is mixed with enriched substance A
Close, obtain enriched substance C；
(2) adding aqueous ammonium chloride solution in enriched substance C, described aqueous ammonium chloride solution is with rich
The volume ratio of collection thing C is 1:2, and the mass concentration of described aqueous ammonium chloride solution is 1.0%, mixing
Rear standing is centrifuged 20 minutes with 1500rpm after half an hour, collects supernatant A respectively with middle
White flock layer A, supernatant A is centrifuged 10 minutes with 1500rpm, collects white bottom it
Layer B, mix with middle white batt layer A, add normal saline, with 1500rpm from
The heart 10 minutes, abandons supernatant B, collects middle white layer C, avoids bottom red during collection
Residual, gleanings is the platelet of enrichment method.
Compared with existing manual PRP technology of preparing, the method have the advantages that
Obtained platelet purity is higher, is conducive to improving its utilization rate and effectiveness.Applicant
Prepared by the platelet having carried out different genera (people, rat, mice), and will the present invention relates to
Method and a classical centrifuging (Anitua method) and secondary centrifuging method (Aghaloo method)
Compare, application automatic blood analyzer (Automated Hematology Analyzer,
XT-2000i, Sysmex Corporation, Japan) result is analyzed.Experimental result table
Bright, compared to a centrifuging and secondary centrifuging method, it is higher that the inventive method can obtain purity
Platelet (people 99.84%, rat 99.19%, mice 99.16%), decrease simultaneously white carefully
The content (people 0.13%, rat 0.81%, mice 0.83%) of born of the same parents, and completely eliminated red
Cell (people 0.00%, rat 0.00%, mice 0.00%), each parameter be superior to once from
Heart method and secondary centrifuging method.The result explanation present invention is applicable to people, rat, three species of mice.
Compared with the product that once centrifugal and secondary centrifuging method obtain, the platelet that the present invention obtains is
Light colour, illustrates that it does not contains the foreign pigment such as erythrocyte.Found by Flow cytometry, this
Inventive method can effectively reduce people, rat, the heteroproteose cell pollution of mice and interference, makes preparation produce
The platelet positive rate of thing is up to more than 99%, hence it is evident that be better than a centrifuging and secondary centrifuging
Method.
The invention provides a kind of highly purified hematoblastic manual preparation method, with existing patent
Authorize or invention disclosed compared bigger difference, the platelet of very high purity can not only be obtained,
Leukocyte and erythrocytic pollution can also be reduced, avoid the untoward reaction that blood plasma causes simultaneously, and
There is higher repeatability and operability, can apply to the experimentation of bone tissue engineer and face
In Chuan.The present invention is expected to become a kind of platelet rich skill that those skilled in the art urgently expect
Art.
Accompanying drawing illustrates:
The preparation of Fig. 1 distinct methods is enriched with hematoblastic pictorial diagram.
Fig. 2. the streaming result of distinct methods preparation enrichment human blood platelets.
Fig. 3. the streaming result of distinct methods preparation enrichment rat platelet.
The streaming result of Fig. 4 distinct methods preparation enrichment mouse platelets.
Detailed description of the invention:
The present invention can be conducted further description and illustrate by the following examples.So
And, protection scope of the present invention is not limited to following embodiment.
Embodiment 1:
(1) in the collecting whole blood of people to the vacuum test tube containing sodium citrate, sodium citrate matter
Amount concentration is 3.2%～3.8%.Anticoagulant is 1:9 with the volume ratio of whole blood.Gather 3mL
Whole blood.
(2) blood and anticoagulant are fully mixed, it is to avoid blood coagulation occurs.
(3) 3mL is contained anticoagulant blood and be assigned to centrifuge tube, be centrifuged with rotating speed 1000rpm
10 minutes, centrifugal rear blood should be divided into three layers: the bottom is erythrocyte, and the superiors are
Platelet poor layer, intermediate layer is rich platelet layer, containing platelet and leukocyte.
(4) utilize pipettor that the superiors' part and mid layer section are extracted out and transfer to new from
In heart pipe, need not deliberately avoid extracting erythrocyte, obtain enriched substance A.
(5) erythrocytic former centrifuge tube adds 1mL normal saline, with rotating speed toward containing remaining
1000rpm is centrifuged 10 minutes, utilizes pipettor that part more than red blood cell layer is extracted out,
Obtain enriched substance B and mix with front enriched substance A once collected, obtaining enriched substance C.
(6) in the centrifuge tube have collected enriched substance C, erythrocyte cracked liquid, described erythrocyte are added
Lysate is the aqueous ammonium chloride solution of mass fraction 1.0%, aqueous ammonium chloride solution and enrichment
The volume ratio of thing C is 1:2, after mixing stand 30 minutes, with rotating speed 1500rpm from
The heart 20 minutes, utilizes pipettor to collect supernatant and middle white batt layer respectively, respectively
It is placed in two new centrifuge tubes.Wherein supernatant is centrifuged 10 minutes with 1500rpm again,
White precipitate bottom collection, mixes with the white flock layer in another pipe, adds 1mL
Normal saline, is centrifuged 10 minutes with rotating speed 1500rpm, collects middle white layer, i.e.
For Platelet Concentrate.
Take rat, Mouse whole blood carries out enrichment method according to above-mentioned steps (1)～(6),
Respectively obtain rat Platelet Concentrate, mice Platelet Concentrate.Each extraction 100 microlitre samples,
Utilizing automatic blood analyzer to detect, repeat experiment, statistics acquired results see table 1.
Take people, rat, Mouse whole blood the most respectively, according to an i.e. Anitua of centrifuging
Method extracts platelet, and concrete grammar is: added by whole blood in the 5ml centrifuge tube containing anticoagulant,
Being centrifuged 6 minutes with 160g, upper strata is PPP, and lower floor is erythrocyte, and two-layer intersection is visible
One the thinnest pale yellow chromatograph, i.e. PRP layer；Discard upper strata (PPP) 1ml, on sucked away
Layer blood plasma, to lower floor's (red blood cell layer) 1～2mm, obtains PRP (Anitua, Int J Oral
Maxillofac Implants,1999,14(4):529-535.)。
Take people, rat, Mouse whole blood, carry according to secondary centrifuging method (Landesberg method)
Taking platelet, concrete grammar is: added by whole blood in the 5ml centrifuge tube containing anticoagulant, with
200g is centrifuged 10 minutes, suction pipe draw whole supernatant (PPP+PRP) to 3mm under interface,
Moving to another centrifuge tube, after balance, second time is centrifuged 10 minutes with 200g, draws on about 3/4
Clear liquid (PPP) is discarded, and residue shakes up and is PRP (Landesberg et al., J Oral
Maxillofac Surg,2000,58(3):297-300)。
The Platelet Concentrate that centrifuging and secondary centrifuging method prepare is also with automatically
Blood analyser detects, and acquired results see table 1.
The table 1. automated blood analysis result to platelet difference preparation method
Table 1 data are the percentage composition of cell quantity.
Table 1 result shows, the inventive method can obtain the higher platelet of purity (people 99.84%,
Rat 99.19%, mice 99.16%), decrease simultaneously leukocyte content (people 0.13%,
Rat 0.81%, mice 0.83%), and (people 0.00%, rat to completely eliminated erythrocyte
0.00%, mice 0.00%), each parameter is superior to a centrifuging and secondary centrifuging method.And
And the inventive method is applicable to humans and animals.
The inventive method, centrifuging, a secondary centrifuging method are being carried out for the first time for human blood
Photo after Li Xin and obtained Platelet Concentrate end-product photo are as shown in Figure 1.With one
The secondary centrifugal product obtained with secondary centrifuging method is compared, and the platelet that the present invention obtains is transparent
Color, illustrates that it does not contains the foreign pigment such as erythrocyte.
Human blood platelets, rat serum that the present invention, centrifuging, secondary centrifuging method are extracted are little
Plate, mouse platelets carry out Flow cytometry respectively, acquired results see respectively Fig. 2,3,
4, result shows, the inventive method can effectively reduce in the platelet of people, rat, mice
Heteroproteose cell pollutes and interference, makes the platelet positive rate preparing product be up to more than 99%, hence it is evident that
It is better than a centrifuging and secondary centrifuging method.
Embodiment 2: prepare Platelet Concentrate
Operating procedure with embodiment 1, except that, in step (6), erythrocyte splitting
Liquid is lymphocyte separation medium (P8610-200, Suo Laibao bio tech ltd, Shanghai),
Lymphocyte separation medium is 1:1 with the volume ratio of enriched substance C.
The people for preparing, rat, mouse platelets purity are more than 99%.
Embodiment 3: prepare Platelet Concentrate
Operating procedure with embodiment 1, except that, step (1) uses heparin
(15IU/mL) being anticoagulant, anticoagulant and volume of whole blood are than for 1:9.
The people for preparing, rat, mouse platelets purity are more than 99%.
Embodiment 4: prepare Platelet Concentrate
Operating procedure with embodiment 1, except that, step (1) uses heparin
(15IU/mL) being anticoagulant, anticoagulant and volume of whole blood are than for 1:9.
In step (6), erythrocyte cracked liquid is lymphocyte separation medium, separation of lymphocytes
Liquid is 1:1 with preparing intermediate product volume ratio.
The people for preparing, rat, mouse platelets purity are more than 99%.
Embodiment 5: prepare Platelet Concentrate
Operating procedure with embodiment 1, except that, step (1) uses hirudin
(0.2mg/mL) being anticoagulant, anticoagulant and volume of whole blood are than for 1:9.
The people for preparing, rat, mouse platelets purity are more than 99%.
Embodiment 6: prepare Platelet Concentrate
Operating procedure with embodiment 1, except that, step (1) uses hirudin be
Anticoagulant, anticoagulant and volume of whole blood are than for 1:9.
In step (6), erythrocyte cracked liquid is lymphocyte separation medium, separation of lymphocytes
Liquid is 1:1 with preparing intermediate product volume ratio.
The people for preparing, rat, mouse platelets purity are more than 99%.
Claims (10)
1. the hematoblastic method of enriching and purifying, it is characterised in that said method comprising the steps of:
(1) whole blood adds anticoagulant, be centrifuged 10 minutes with 1000rpm, after centrifugal end,
Draw red blood cell layer above section, obtain enriched substance A；Residue red blood cell layer adds
Normal saline, then be centrifuged 10 minutes with 1000rpm, take red blood cell layer above section,
Obtaining enriched substance B, enriched substance B mixes with enriched substance A, obtains enriched substance C；
(2) in enriched substance C, add erythrocyte cracked liquid, stand after mixing after half an hour with
1500rpm is centrifuged 20 minutes, collects supernatant A and middle white batt layer A respectively,
Supernatant A is centrifuged 10 minutes with 1500rpm, collects white layer B bottom it, with
Middle white batt layer A mixes, and adds normal saline, is centrifuged 10 with 1500rpm
Minute, abandon supernatant B, collect middle white layer C, during collection, avoid bottom red
Residual, gleanings is the platelet of enrichment method.
2. the method for claim 1, it is characterised in that in described step (1), described anti-
Solidifying agent is sodium citrate, heparin or hirudin.
3. the method for claim 1, it is characterised in that in described step (2), described red
Cell pyrolysis liquid is aqueous ammonium chloride solution or lymphocyte separation medium.
4. method as claimed in claim 3, it is characterised in that in described step (2), described chlorine
The volume ratio changing aqueous ammonium and enriched substance C is 1:2, and the quality of described aqueous ammonium chloride solution is dense
Degree is 1.0%.
5. method as claimed in claim 3, it is characterised in that in described step (2), used
Lymphocyte separation medium is purchased from Suo Laibao bio tech ltd, Shanghai, lymphocyte separation medium
It is 1:1 with the volume ratio of enriched substance C.
6. method as claimed in claim 2, it is characterised in that described anticoagulant and the volume of whole blood
Ratio is 1:9.
7. method as claimed in claim 2, it is characterised in that described sodium citrate mass concentration is
3.2%～3.8%.
8. the method for claim 1, it is characterised in that in described step (2), collect it
Bottom white layer B, mixes with middle white batt layer A, adds normal saline, described life
Addition is volume of whole blood 0.2～1 times of reason saline.
9. the method for claim 1, it is characterised in that in described step (1), remain red
Adding normal saline in cellular layer, the addition of described normal saline is the 0.2～1 of volume of whole blood
Times.
10. the method for claim 1, it is characterised in that described method sequentially includes the following steps:
(1) adding anticoagulant in whole blood, anticoagulant is 1:9 with the volume ratio of whole blood, with 1000rpm
Centrifugal 10 minutes, after centrifugal end, draw red blood cell layer above section, obtain enriched substance A；
Residue red blood cell layer adds normal saline, then is centrifuged 10 minutes with 1000rpm, take red carefully
Born of the same parents' layer above section, obtains enriched substance B, and enriched substance B mixes with enriched substance A, obtains richness
Collection thing C；
(2) in enriched substance C, aqueous ammonium chloride solution, described aqueous ammonium chloride solution and enriched substance are added
The volume ratio of C is 1:2, and the mass concentration of described aqueous ammonium chloride solution is 1.0%, quiet after mixing
It is centrifuged 20 minutes with 1500rpm after putting half an hour, collects supernatant A and middle white respectively
Batt layer A, supernatant A is centrifuged 10 minutes with 1500rpm, collects white layer B bottom it,
Mix with middle white batt layer A, add normal saline, be centrifuged 10 points with 1500rpm
Clock, abandons supernatant B, collects middle white layer C, avoids the red residual in bottom, receive during collection
Collection thing is the platelet of enrichment method.
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| CN107384856A (en) * | 2017-07-28 | 2017-11-24 | 重庆赛纳思生物科技有限公司 | A kind of method for preparing platelet lysates liquid |
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| CN112237755B (en) * | 2019-07-18 | 2023-12-05 | 北京纳通医学科技研究院有限公司 | Preparation method and preparation device of platelet-rich plasma and prepared platelet-rich plasma |
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| CN111388649B (en) * | 2020-03-19 | 2023-09-12 | 杭州三江上御生物科技有限公司 | Platelet-rich plasma placenta polypeptide composition and application thereof |
| CN114015641A (en) * | 2021-07-13 | 2022-02-08 | 安徽农业大学 | Porcine platelet lysate culture medium and application thereof |
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